Mitotic Chromosome Binding Predicts Transcription Factor Properties in Interphase

bioRxiv preprint doi: https://doi.org/10.1101/404723; this version posted August 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Mitotic chromosome binding predicts transcription factor properties in interphase Authors: Mahé Raccaud1, Andrea B. Alber1,2, Elias T. Friman1,2, Harsha Agarwal3, Cédric Deluz1, Timo Kuhn3, J. Christof M. Gebhardt3 and David M. Suter1,4 Affiliations: 1Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland 2These authors contributed equally 3Institute of Biophysics, Ulm University, Albert-Einstein-Allee 11, Ulm 89081, Germany 4Lead contact Corresponding author: David M. Suter ([email protected]) Summary Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence- specific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape. Keywords: transcription factors; mitotic chromosome binding; non-specific DNA binding; specific DNA binding; transcription factor search efficiency; transcription factor occupancy; chromatin accessibility 1 bioRxiv preprint doi: https://doi.org/10.1101/404723; this version posted August 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Transcription factors (TFs) are central players in the regulation of gene expression. Each TF binds to specific regulatory sequences to regulate transcription of target genes. The ability of TFs to occupy their specific sites in the genome depends on their nuclear concentration, their ability to search the genome and the chromatin environment of their binding sites. TFs search the genome both by 3D diffusion and by facilitated diffusion using local 1D search mediated by sliding, hopping and intersegment transfer (Dror et al., 2016; Hu et al., 2008; Marklund et al., 2013; Subekti et al., 2017; Takayama and Clore, 2011; Vuzman et al., 2010). These local interactions can strongly modulate search efficiency and mainly depend on transient non-specific protein-DNA association (Berg et al., 1981; Hettich and Gebhardt, 2018; von Hippel and Berg, 1989; von Hippel et al., 1974; Winter et al., 1981), which are essentially mediated by electrostatic interactions (Matthew and Ohlendorf, 1985; Takeda et al., 1986; Kenar et al., 1995; Kalodimos et al., 2004; Barbi and Paillusson, 2013; Vuzman and Levy, 2010; Desjardins et al., 2016; Vo et al., 2017). However, non- specific DNA binding of most TFs remains uncharacterized, and thus to which extent this property impacts genome-wide occupancy of TFs is unknown. A minority of TFs were shown to physically associate with mitotic chromosomes (Raccaud and Suter, 2017). These interactions can be identified by ChIP-seq on purified mitotic cells and co- localization analysis of TFs with mitotic DNA using fluorescence microscopy. While ChIP-seq essentially identifies sequence-specific DNA binding, fluorescence microscopy allows to quantify the association of TFs with mitotic chromosomes independently of their enrichment on specific genomic sites (Raccaud and Suter, 2017). Both non-specific and specific DNA binding of TFs to mitotic chromosomes have been described. However, the often small number of specifically-bound loci on mitotic chromosomes (Caravaca et al., 2013; Deluz et al., 2016; Festuccia et al., 2018; Kadauke et al., 2012), the mild or null sensitivity to alterations of specific DNA binding properties (Caravaca et al., 2013; Festuccia et al., 2016), and the absence of quantitative relationship between mitotic ChIP- seq datasets and fluorescence microscopy (Festuccia et al., 2018) suggest that the co-localization of TFs with mitotic chromosomes observed by microscopy is mainly due to non-specific DNA interactions. Converging evidence from the literature further corroborates this view. SOX2 and FOXA1 are strongly associated with mitotic chromosomes (Caravaca et al., 2013; Deluz et al., 2016), and these also display high non-specific affinity for DNA in vitro (Sekiya et al., 2009; Soufi et al., 2015). In contrast, OCT4 displays less visible association with mitotic chromosomes (Deluz et al., 2016), and has low non-specific affinity for DNA in vitro (Soufi et al., 2015). Finally, FOXA1 mutants with decreased non-specific DNA affinity but retaining their specificity for the FOXA1 motif also display reduced mitotic chromosome association (Caravaca et al., 2013). Many TFs binding to mitotic chromosomes also display pioneer properties (Caravaca et al., 2013; Kadauke et al., 2012; Soufi et al., 2012; Zaret, 2014), i.e. they are able to bind and open condensed 2 bioRxiv preprint doi: https://doi.org/10.1101/404723; this version posted August 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. chromatin regions, allowing them to rewire gene expression programs to mediate cell fate decisions. However, the existence of a common molecular mechanism underlying mitotic chromosome binding and pioneer activity remains uncertain. Here we measured mitotic chromosome binding (MCB) of 502 mouse TFs in live mouse embryonic stem (ES) cells. We show that MCB is predictive of interphase TF properties such as sub-nuclear localization, mobility, and of the large differences in TF ability to occupy specific genomic sites. We propose that the co-localization of TFs with mitotic chromosomes is a proxy for TF non-specific DNA binding properties, which regulate TF search efficiency for their specific binding sites and thereby their impact on chromatin accessibility. Results Large-scale quantification of TF association to mitotic chromosomes To measure mitotic chromosome binding for a large number of TFs, we constructed a doxycycline (dox)-inducible lentiviral vector library of 757 mouse TFs fused to a yellow fluorescent protein (YPet) (Figure 1A). This library was used to generate a corresponding library of mouse embryonic stem (ES) cell lines to quantify TF association to mitotic chromosomes by live cell fluorescence microscopy. One day before imaging, cells were seeded in 96-well plates and treated with dox to induce the expression of TF-YPet fusion proteins. Cells were maintained in ES cell proliferation medium and imaged by wide-field fluorescence microscopy. We used a semi-automated pipeline to detect cells in metaphase, which allows for easy quantification of mitotic chromosome binding since chromosomes are most spatially confined in this phase. Of note, we did not observe any obvious differences in co-localization of TFs with mitotic chromosomes between prophase, metaphase and anaphase. We used the Mitotic Bound Fraction (MBF) as a metric for mitotic chromosome binding, defined as the averaged YPet fluorescence intensity on metaphase chromosomes multiplied by the fraction of cellular volume occupied by DNA (as measured by confocal microscopy, see STAR Methods), divided by the total YPet signal (Figure 1A). The reliability of wide-field fluorescence measurements was confirmed by their correlation with those performed by confocal microscopy (Figure S1A-B, Table S1). In total 502 TFs yielded a sufficiently strong fluorescent signal in metaphase to allow measuring their MBF, and for 94% of these we could measure the MBF in at least 10 cells (see STAR methods). We defined three bins of TFs based on visual inspection of the YPet signal in the area occupied by metaphase chromosomes: depleted (YPet signal lower than in the cytoplasm), intermediate (YPet signal equal to that of the cytoplasm) or enriched (YPet signal higher than in the cytoplasm), which corresponded to MBFs < 16.5%, 16.5-23% and >23%, respectively. 24% of TFs fell in the “depleted” bin, 54% in the “intermediate” bin, and 22% in the “enriched” bin (Figure 1B). Most TFs previously reported to be highly enriched on mitotic chromosomes and present in our library, such as FOXA1 (Caravaca et al., 2013), GATA1 (Kadauke 3 bioRxiv preprint doi: https://doi.org/10.1101/404723; this version posted August 31, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. et al., 2012), GATA4 (Caravaca et al., 2013), SOX2 (Deluz et al., 2016; Teves et al., 2016), RUNX2 (Young et al., 2007), ESRRB (Festuccia et al., 2016), RBPJ (Lake et al., 2014) and HNF1b (Verdeguer et al., 2010), fell in the “enriched” category (Table S2), suggesting that

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